Tuberculosis, one of the great scourges of mankind, is reemerging as a significant health problem in the U.S., most frequently afflicting the elderly, the homeless, individuals with AIDS, and immigrants from nations where the incidence of TB is high. Although the causative agent of TB, Mycobacterium tuberculosis, was identified a century ago, knowledge about the fundamental physiological capabilities, genetics, specific virulence determinants, and mechanism(s) of pathogenicity of these bacilli is almost nonexistent. We therefore propose to identify and characterize genes from M. tuberculosis that are important in the pathogenicity of this organism through molecular and functional analyses of genomic DNA and recombinant molecules from genomic libraries. Specifically, we will (i) identify regions of chromosomal DNA that re unique to M. tuberculosis H37Rv by subtractive hybridizations between genomic DNAs from H37Rv and H37Ra and between H37Rv and BCG, (ii) prepare complementary DNA (cDNA) libraries from M. tuberculosis H37Rv bacilli purified from human macrophages at different times after Infection and from H37Rv bacilli grown in broth culture, (iii) identify genes that are expressed at different stages of infection by subtractive hybridizations between cDNAs from macrophage- grown and broth-grown M. tuberculosis H37Rv, (iv) subclone genes into appropriate vectors and introduce them into H37Ra or BCG to determine whether or not the cloned genes will enhance survival and/or growth of the avirulent derivatives in macrophages, and (v) characterize the unique regions and genes by nucleotide sequence determination and computer analysis.